PROJECT SUMMARY ? PROJECT 1 Esophageal cancers comprise two major subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Herein, we focus upon the pathogenesis and translational therapeutics of ESCC and draw comparisons to EAC when possible. ESCC is the deadliest of all human squamous cell carcinomas. Its common genetic lesions include the p53 tumor suppressor and epidermal growth factor receptor (EGFR) as revealed in Project 3. Mutant p53 impairs cell-cycle arrest or apoptosis in response to cellular stress. EGFR activates mitogen-activated protein kinase (MAPK). Additionally, EGFR and mutant p53 facilitate epithelial-mesenchymal transition (EMT). EMT fosters intra-tumoral heterogeneity and tumor stroma remodeling in ESCC. The malignant properties of ESCC may stem from a subset of cells characterized by high CD44 expression (=CD44H) that in turn promote tumor invasion in the stroma, tumor metastasis (e.g. lung) and conventional therapeutic resistance. The overall objective in Project 1 is to elucidate the molecular mechanisms by which mutant p53 and the tumor microenvironmental cues cooperate to drive ESCC invasion and metastasis, and as a result, unravel new therapeutic targets in synergy with Projects 2 and 3. Utilizing cell lineage-traceable mice treated with the esophageal carcinogen 4-nitroquinoline 1-oxide (4NQO), three- dimensional (3D) culture systems that recapitulate human pathology, and patient-derived xenograft (PDX) tumors with clinically available pharmacological inhibitors, our published and unpublished preliminary data indicate that the interplay between ESCC cells (harboring mutant p53) and cancer-associated fibroblasts (CAFs) and ESCC cells triggers EMT to induce CD44H cells. CD44H cells produce cytokines RANTES and interleukin (IL)-6 to drive ESCC invasion. CD44H cells also display downregulation of the Rab GTPase- coupling protein (RCP)-mediated endocytosis recycling pathway to stabilize cell surface proteins, including EGFR and the RANTES receptor CCR5. Moreover, mutant p53 (p53R172H) facilitates ESCC EMT and lung metastasis. Our central hypothesis is that mutant p53 and CAFs foster the emergence of CD44H ESCC cells with increased malignant properties and possible metastasis. We will test our hypothesis by pursuing the following interrelated Specific Aims: (1) To elucidate the role of RANTES as a mediator of the crosstalk between ESCC cells and CAFs; (2) To delineate how mutant p53 facilitates metastasis via CD44H cells; and (3) To determine how mutant p53 impacts upon the endocytic recycling pathway in ESCC invasion. We will work closely with Projects 2 and 3, with support from the Core Facilities, to translate our findings into new therapeutics that disable the protumorigenic ESCC microenvironment and subsequent proclivity for metastasis.